Viruses in Biotechnology - Institute of Food and Agricultural ...
Viruses in Biotechnology Dr. James Maruniak Animal Viruses - Vaccines Mutated viruses- generated by recombinant DNA techniques Vaccine vectors- Yellow fever chimeric vaccines Other ENV gene Vaccinia used for
YFV Cytokine expression- to boost immunity Subunit vaccines- envelope proteins from other viruses Problems with low antigenicity- add enhancers or try other promoters Multiple vaccines in it- influenza & Hepatitis B env Disseminated vaccinia- is a problem Foreign gene
Positive control + control For treatment of existing HPV infection, techniques to improve cellular immunity by enhancing viral antigen recognition are being studied. For this purpose, the oncogenic proteins E6 and E7 of HPV-16 and -18 are the focus of current clinical trials for cervical cancer patients. Clinical Pharmacology & Therapeutics (2007) 81, 259264. GARDASIL: Prophylactic Human Papillomavirus Vaccine Development
From Bench Top to Bed-side L. Shi, H. L. Sings, J. T. Bryan, B. Wang, Y. Wang, H. Mach, M. Kosinski, M. W. Washabaugh, R. Sitrin and E. Barr Merck Research Laboratories, West Point, Pennsylvania, USA Abstract GARDASIL (Merck, Whitehouse Station, NJ) is a non-infectious recombinant, quadrivalent vaccine prepared from the highly purified virus-like particles (VLPs) of the major capsid proteins of human papillomavirus (HPV) types 6, 11, 16, and 18. GARDASIL is the first vaccine approved for use in women aged 926 years for the prevention of cervical cancer and genital warts, as well as
vulvar and vaginal precancerous lesions. This report describes some of the key preclinical efforts, achievements in pharmaceutical development, in vivo animal evaluation, and clinical trial data. Title: Papillomavirus vaccine Document Type and Number: United States Patent 7169585 Abstract: A method of providing papillomavirus like particles which may be used for diagnostic purposes or for incorporation in a vaccine for use in relation to infections caused by papillomavirus. The method includes an initial step of constructing one or more recombinant
DNA molecules which each encode papillomavirus L1 protein or a combination of papillomavirus L1 protein and papillomavirus L2 protein followed by a further step of transfecting a suitable host cell with one or more of the recombinant DNA molecules so that virus like particles (VLPs) are produced within the cell after expression of the L1 or combination of L1 and L2 proteins. The VLPs are also claimed per se as well as vaccines incorporating the VLPs. Virus-like particles (VLPs) assembled from the L1 protein
of Human Papillomavirus 16 HPV-assembly. Five L1 main structure proteins (monomers) are assembling to one pentamer (capsomere). Finally, 72 capsomeres
are building one viruslike particle (VLP) (according to Lutz Gissmann, modified). The HPV major capsid protein L1 can fold correctly and self-assemble into VLPs when expressed in eukaryotic cells. VLPs aim to protect against the development of cervical cancer; protection would be mediated by the induction of high titres of neutralizing
antibodies against the HPV genotypes in the vaccine that prevent the virus infecting the transformation zone, a metaplastic area between the squamous and columnar epithelia in the cervix, where most cancers arise. Delivering on the promise: HPV vaccines and cervical cancer John T. Schiller & Philip Davies Nature Reviews Microbiology 2, 343-347 (April 2004)
HPV L1-VLP specific IgM, IgA, IgG1 and IgG4 antibody titers. Sera from rhesus macaques immunized at week 0, 8 and 24 with 2 :g each of HPV 6, 11, 16 and 18 L1-VLPs formulated with Merck Aluminum Adjuvant (+MAA) (- -) or 2 g each of HPV 6, -) or 2 :g each of HPV 6, 11, 16 and 18L1-VLPs alone (-MAA) (- -) were collected at the indicated time points and tested for HPV L1 VLP specific (A) IgM, (B) IgA, (C) IgG1 and (D) IgG4 titers in a multiplexed detection assay. Responses are reported as GMTs (n = 5 monkeys per group) for HPV 16. Arrows indicate vaccination boosts at weeks 8 and 24. Graphs shown are for HPV 16 and are representative of HPV 6, 11, and 18. The Journal of Infectious Diseases
A Phase 1 Study of a Recombinant Virus-like Particle Vaccine against Human Papillomavirus Type 11 in Healthy Adult Volunteers Thomas G. Evans,1,a William Bonnez,1 Robert C. Rose,1,2 Scott Koenig,3 Lisa Demeter,1,2 JoAnn A. Suzich,3 Diane O'Brien,1 Meredith Campbell,1 Wendy I. White,3 James Balsley,3 and Richard C. Reichman1,2 1Infectious Diseases Unit, Department of Medicine, and 2Department of Microbiology and
Immunology, University of Rochester School of Medicine and Dentistry, Rochester, New York; 3MedImmune, Gaithersburg, Maryland Received 17 March 2000; revised 31 January 2001; electronically published 24 April 2001. Virus-like particles (VLPs) produced from the L1 protein of several papillomaviruses have induced protection from infection after live challenge in animal models. In the present study, the safety and immunogenicity of a human papillomavirus (HPV)11 L1 VLP candidate vaccine were measured in a phase 1, dose-finding trial in humans. The vaccine was well tolerated and induced high levels of both binding and neutralizing antibodies. Marked increases in lymphoproliferation to HPV-11 L1 antigens were noted after the second vaccination. In addition, lymphoproliferation was induced after vaccination in peripheral blood
mononuclear cells (PBMC) stimulated with heterologous L1 VLP antigens of HPV types 6 and 16. Statistically significant increases in HPV antigen specific interferon and interleukin-5 production were measured from PBMC culture supernatants. This candidate HPV VLP vaccine induced robust B and T cell responses, and T cell helper epitopes appear to be conserved across HPV types. L1 VLPs corresponding to HPV-6, -11, -16, and -18 and to parvovirus B19 VP2 were produced at MedImmune, using the baculovirus expression system. Purification was accomplished by cesium chloride and sucrose gradient centrifugation and by phenyl hydrophobic interaction chromatography. The final bulk product was dialyzed and sterilized by filtration. A Controlled Trial of a Human Papillomavirus Type 16 Vaccine
Laura A. Koutsky, Ph.D., Kevin A. Ault, M.D., Cosette M. Wheeler, Ph.D., Darron R. Brown, M.D., Eliav Barr, M.D., Frances B. Alvarez, R.N., Lisa M. Chiacchierini, Ph.D., Kathrin U. Jansen, Ph.D., for the Proof of Principle Study Investigators Background Approximately 20 percent of adults become infected with human papillomavirus type 16 (HPV16). Although most infections are benign, some progress to anogenital cancer. A vaccine that reduces the incidence of HPV-16 infection may provide important public health benefits. Methods In this double-blind study, we randomly assigned 2392 young women (defined as females 16 to 23 years of age) to receive three doses of placebo or HPV-16 virus-likeparticle vaccine (40 g per dose), given at day 0, month 2, and month 6. Genital samples to test for HPV-16 DNA were obtained at enrollment, one month after the third vaccination, and every six months thereafter. Women were referred for colposcopy according to a protocol. Biopsy tissue was evaluated for cervical intraepithelial neoplasia and analyzed for HPV-16 DNA with use of the polymerase chain reaction. The
primary end point was persistent HPV-16 infection, defined as the detection of HPV-16 DNA in samples obtained at two or more visits. The primary analysis was limited to women who were negative for HPV-16 DNA and HPV-16 antibodies at enrollment and HPV-16 DNA at month 7. Results The women were followed for a median of 17.4 months after completing the vaccination regimen. The incidence of persistent HPV-16 infection was 3.8 per 100 woman-years at risk in the placebo group and 0 per 100 woman-years at risk in the vaccine group (100 percent efficacy; 95 percent confidence interval, 90 to 100; P<0.001). All nine cases of HPV-16related cervical intraepithelial neoplasia occurred among the placebo recipients. Administration of this HPV-16 vaccine reduced the incidence of both HPV-16 infection and HPV-16 related cervical intraepithelial neoplasia. Immunizing HPV-16negative women may eventually reduce the incidence of cervical cancer.
Vaccine Promising Against Disease-Causing HPV NEW YORK (Reuters Health) - April 7, 2005 Certain strains of the virus responsible for causing genital warts -- human papillomavirus or HPV -are also likely to trigger cervical cancer. Now comes news that a vaccine designed to combat four of these strains can dramatically reduce persistent infection and the risk of developing warts and cancer. The findings are similar to results seen when a two-hit vaccine was tested in North America and Brazil last year. However, in that study, the vaccine targeted only HPV-16 and HPV-18, the main cancer-causing types, whereas the new four-strain 'quadrivalent' vaccine targets these types as well as HPV-6 and HPV-11, the ones most often linked to genital warts. The study involved 552 young women who were not pregnant, had no history of abnormal Pap
smears, and had had no more than four sexual partners. Women with previous HPV infection were not excluded from the study. The women were given three shots of the quadrivalent vaccine or placebo injections over a period of six months. They were then followed for 36 months with regular gynecologic exams, cervical testing for HPV, and Pap smear tests. Compared with placebo injections, the vaccine led to 90 percent fewer cases of persistent infection or disease due to HPV types 6, 11, 16, or 18, lead study author Dr. Luisa L. Villa, from the Ludwig Institute for Cancer Research in Sao Paulo, Brazil, and colleagues report in the medical journal Lancet Oncology. The vaccine was 89 percent effective in preventing infection with the four HPV types, and 100 percent effective in preventing the diseases associated with these types.
The study was funded by Merck Research Laboratories, which is developing the vaccine. SOURCE: Lancet Oncology, online April 7, 2005. Expert Opin Emerg Drugs. 2005 Feb;10(1):5-19. Emerging human papillomavirus vaccines. Christensen ND. The Pennsylvania State University College of Medicine, The Department of Microbiology and Immunology, Hershey, PA 17033, USA. Human papillomavirus (HPV) infections are a leading cause of virus-associated cancers of the anogenital, oropharyneal and cutaneous epithelium. The most prevalent of these is cervical cancer, which is responsible for approximately 500,000 deaths annually worldwide. A group of about 15
serologically unrelated 'high-risk' HPV types are responsible for almost all HPV-associated cancers. Prevention of papillomavirus infection can be achieved by induction of capsid-specific neutralising antibodies in preclinical animal papillomavirus models and in recent human clinical trials. High titres of conformationally-dependent, type-specific HPV-neutralising antibodies are triggered by HPV virus-like particle (VLP) vaccines. Overcoming the problems of type-specificity of the responses to these VLP vaccines is a potentially important area of current HPV vaccine research, with an emphasis on induction of more broadly cross-protective neutralising responses. Viral oncogenes E6 and E7 are continuously present in HPV-associated cancers and are prime targets for HPV therapeutic vaccines. A variety of approaches are being tested in therapeutic vaccine clinical trials and in various preclinical animal papillomavirus models for efficacy. Approaches include genetic vaccines, recombinant virus vaccines, dendritic cell-based strategies,
immunomodulatory strategies and various combination strategies to maximise cell-mediated immunity to papillomavirus proteins present in HPV infections and cancers. The success of preventive HPV VLP vaccines in clinical trials is clear. However, current therapeutic vaccine trials are less effective with respect to disease clearance. Animal Viruses - Vaccines Avipoxvirus Results in abortive virus infection in humans Not highly immunogenic, must use multiple times
Canarypox - attenuated and used for rabies Canarypox expresses measles glycoproteins Entomopoxvirus Abortive infection in mammalian cells can be engineered to transiently express proteins Animal Viruses - Vaccines Poliovirus Sabin vaccine attenuated live virus has been engineered to express foreign proteins
Insertion of large foreign DNA into VP1 to make antigen on virus surface, but requires helper poliovirus Insert small DNA (60 bp) in loop region of VP1 to express epitope in highly immunogenic region on virion Foreign antigens can be part of the poliovirus polyprotein that is cleaved Animal Viruses - Vaccines DNA vaccines
Stimulate humoral and cell-mediated immunity May not prime deleterious immune response due to incoming virus proteins Bypasses toxic effect of virion proteins such as: ??? * Pcmv=cytomegalovirus promoter, pA=polyadenylation signal, f1 ori & ColE1=bacterial origins of replication, SV40=promoter, amp=ampicillin resistance,
RSV LTR=Rous sarcoma virus promoter, plus the indicated restriction sites P CMV=cytomegalovirus promoter, pA=polyadenylation signal, oriP & ColE1=bacterial origins of replication, SV40=promoter, amp=ampicillin resistance, pRSV=Rous sarcoma virus promoter, EBNA=Epstein Barr Virus Nuclear Antigen, PTK= Thymidine Kinase promoter, plus the indicated restriction sites The Making of a DNA Vaccine Against West Nile Virus http://www3.niaid.nih.gov/news/newsreleases/2005/wnvgraphic.htm
Comparison of Viral-Based Vectors for Vaccine Applications PARENT VIRUS Retrovirus Adenovirus Nucleic Acid AAV VEE
RNA-> DNA DNA DNA RNA YES YES
YES NO MEDIUM MEDIUM MEDIUM
HIGH Lymph Node Targeting NO NO NO YES
Pre-existing Vector-specific Immune Response NO YES YES NO
Expression Level Induction of Ab, CTL, and Mucosal Immune Responses Tested in Human Clinical Trials Ab, CTL, and Mucosal * Live attenuated VEE vaccine strain TC83 used extensively in humans.
Ab, CTL, and Mucosal YES* Animal Viruses Diagnostic Virology make reagents To test for recent infections for antibodies Genomes in polymerase chain reaction for diagnostics Expression vectors for protein production
Cytomegalovirus constitutive promoters SV40 promoters and signal sequences used Adenovirus genes such as E1A Make viral proteins for diagnostic assays Protein or Pharmaceutical development Develop heart disease drugs against Creactive protein induced by Herpesvirus
cultured in the laboratory for this assay Kaposi's sarcoma linked with a herpesvirus. Use virus to test drugs. Brain tumors targeted with engineered adenovirus Genital herpes need new antiviral drugs tested Evaluation of a test based on baculovirus expressed glycoprotein G for detection of herpes simplex virus type specific antibodies. Division of Viral and Rickettsial Diseases, Centers for Disease Control, Atlanta, Georgia 30333.
J Infect Dis. 1991 Dec;164:1196-9. An immunoblot assay for discrimination of antibodies to herpes simplex virus (HSV) types 1 and 2 was devised using extracts of recombinantbaculovirus-infected insect cells expressing HSV-1 or -2 glycoprotein G (gG1 or gG2). The assay was evaluated by comparing its results with those obtained by using an immunodot assay based on gG immunopurified from HSV-1- and HSV-2-infected cells. The assay is accurate and reproducible. The ease of antigen production should allow the test to become widely available Viruses used in Biological
Control BACULOVIRUS EPA Registered wt Baculoviruses in USA Autographa californica MNPV vegetable insects Spodoptera exigua MNPV - cotton and vegetable caterpillars Neodiprion sertifer SNPV forest caterpillars Lymantria dispar MNPV forest caterpillars Cydia pomonella GV fruit caterpillars
Helicoverpa zea SNPV - cotton and corn insects Orgyia pseudotsugata MNPV - forest caterpillars After recombination with the baculovirus genome and insect cell infection P ph= polyhedrin promoter, P p10= promoter, ph pA=polyadenylation signal, Recombination sequences from part of the baculovirus genome, ColE1=plasmid origin of replication in
bacteria, Amp=ampicillin resistance for protein expression after recombination with the baculovirus genome and insect cell infection P ph= polyhedrin promoter, PETL= promoter, Recombination sequences from part of the baculovirus genome, ColE1=plasmid origin of replication in bacteria, 5 lacZ Fragment = galactosidase gene for recombination in Baculovirus
genome, (His)6= 6 histidine residues for nickel column purification of fused protein pGenBac2.1 is a transfer vector for high level eukaryotic expression of recombinant proteins in insect cells. This vector is compatible with Invitrogen Bac-to-Bac Baculovirus Expression System. The gene of interest can be easily cloned downstream of polyhedron promoter. This transfer vector recombines with a parent bacmid in E. coli cells to form an expression bacmid. High level gene expression is achieved by infecting insect cell lines with the recombinant bacmid. A simplified schematic of the capabilities for glycosylation in most lepidopteran cell lines. Note that there is evidence that glycosidases do some trimming of these structures, particularly the GlcNAc residues from the intermediate structure.
Baculovirus wild type and recombinant plaques Protein Sciences collaborates with some of the largest pharmaceutical and biopharmaceutical firms in the world, as well as with (virtual) biotech companies, and academic and government institutions. A selection of our customers:
Rabbit Calicivirus Behind CMV promoter CaMV 35S is Cauliflower Mosaic Virus promoter used in RNA silencing Tobacco Mosaic Virus in plants Tobacco Mosaic Virus BCI
CaMV 35S Enhancement of freezing tolerance of plants CaMV 35S= Cauliflower Mosaic Virus 35S promoter Genetic Transformation for Virus Resistance Insert DNA sequences isolated from WMV
(watermelon mosaic virus) and other viruses into plant cell cultures Select transformed plants from cell cultures Test plants and progeny for resistance in greenhouse and field Integrate progeny into patented process to develop new seedless varieties Foreign Genes Commonly Used in Transgenic Organisms Plants
Bt (Bacillus thuringiensis) delta endotoxins against lepidoptera, coleoptera herbicide tolerance plant virus resistance Plant Made Antibody Targets Hepatitis B Virus NEW YORK (Reuters Health) - June 23, 2004 Japanese scientists have successfully used genetically engineered cells from the tobacco plant to produce a human antibody that homes in on a molecule on the surface of the hepatitis B virus (HBV). Currently, treatment of HBV may include infusion of serum containing
antibodies, called immunoglobulin, collected from blood donors. The new results demonstrate the feasibility of producing anti-hepatitis antibodies in plants "as an alternative to anti-HBV human immunoglobulins," Dr. Akira Yano from the National Institute of Public Health in Tokyo and colleagues write in the Journal of Medical Virology. "Our plant-derived (antibody) has the potential to be a cheap and effective pharmaceutical" for the prevention and treatment of HBV infection, Yano told Reuters Health. Transgenic Plants in Agriculture Example of companies involved in
Which of the following steps is not used in construction of a vaccinia vaccine vector? 1. cloning of foreign gene 2. transfection of vaccinia genome 3. transfection of recombinant plasmid 4. infection of tissue culture cells 5. homologous recombination
Project Management is a way of doing business for all project team members Provide project managers easy-to-use checklists and templates that target the fundamental concepts of project management Utilization of the tools will increase the probability of project success Provide...
Light Scattering Vijay Natraj Ge152 ... Each mixing group is a combination of 4 aerosol components Lognormal distribution Global Climatology of Aerosol Types Scattering Matrix Describes transformation from incident to viewing direction In many cases, function only of scattering angle...
The copper cycle. Cut and paste the stages in the copper cycle to show how copper can be extracted. How else can copper be extracted? Iron and steel. What is iron? Find the symbol and atomic number. Find out where...
CSI3131 - Lab 1 Observing Process Behaviour Running Linux under Virtual PC Start Virtual PC This Windows program provides a virtual machine to run any other operating system It offers an environment that looks like the raw computer system, that...
Id: Ego: Superego: A SHORT ACTIVITYActivity Handout 12.1. Jake is going on his first date with a really popular girl. He still can't believe that she agreed to go out with him. During the movie they are sitting so close...
the multiplicative group in GF(28) - that's S1. Then apply the affine transformation - that's S2. S2 is the affine transform: - wikipedia. Inverse affine transform. AES algorithm. consider the input byte expressed in hex. the two nibbles provides the...
Adjust Your Attitude. What we tell ourselves about the situation will determine how we feel about it. Looking at the good side of things (having a positive attitude) can help us feel better. Discover Your Choices. In every situation there...